Tocopherols are natural products widely used as vitamin E for human health, cosmetic ingredients and food antioxidants. The main source of tocopherols is the deodorized distillate produced by the manufacture process of edible oil. The main constituents of the tocopherols extracted from deodorized distillate are α, β, γ, and δ-tocopherols. Their molecular structures have a common core ring, differing only in the number of attached methyl groups. Although the structures are quite similar, it is known in the art that α-tocopherol has the highest biopotency among all the isomeric forms. Current research involving tocopherols is focused on α-tocopherol, emphasizing α-tocopherol's use as an antioxidant.
In normal physiological cell function, lysosomal enzymes break down macromolecules in cells. Normal metabolism allows the cells to remove expended metabolites. Defects or deficiencies of lysosomal enzymes or other lysosomal components can result in the accumulation of undegraded metabolites. These defects or deficiencies result in Lysosomal Storage Disorders (LSDs).
Cholesterol is an essential component of the cell membrane and plays an important role in maintaining integrity and fluidity of the cell membrane with additional lipids. The appropriate proportion and makeup of cholesterol in the cell membrane is important for regulation of cell signaling, receptor function, ion permeability and endocytosis. In addition, cholesterol is the precursor molecule for synthesis of bile acids, Vitamin D and steroid hormones. Cholesterol concentration is regulated on two levels; in the body as a whole and intracellularly. Cholesterol is primarily synthesized in cells and transported in blood by lipid binding proteins. While the mechanisms of cholesterol transport in blood and cholesterol synthesis in cells are well characterized, the many details of free cholesterol trafficking in cells remain unclear. Cholesterol enters cells through low-density lipoprotein (LDL) receptors via endocytosis or cholesterol is de novo synthesized inside cells. The internalized cholesterol esters are hydrolyzed by acid lipase to form free cholesterols in late endosomes/lysosomes that are delivered to NPC1 and NPC2 proteins for further processing. Cholesterol may be transported to other proteins and/or to membrane microdomains such as lipid rafts and other vesicles. It is known that most of these free cholesterols recycle back to cell membrane; some of them efflux out of cells, and some of them move to ER for storage after esterification.
Lysosomal storage diseases (lysosomal storage disorders) are a group of ˜50 diseases with a common feature of lysosomal accumulation of macromolecules such as lipids or glycoproteins in patient cells. The diseases are caused by inherited genetic mutations that result in deficiency of lysosomal enzyme or protein. Once the hydrolysis or transport of macromolecules such as lipids and glycoproteins is reduced by a mutation of an enzyme or protein in lysosome, they are accumulated which causes the enlargement of lysososomes as well as malfunction of lipids recycling and utilization in cells. Degeneration or death of affected cells occurs in certain tissues which are varied in different lysosomal storage diseases. The hepatosplenomegaly and neuronal degeneration are common features of many lysosomal storage diseases.
One identified LSD is Niemann-Pick type C disease (NPCD), an autosomal recessive disease with an estimated incidence of 1:150,000. This disease is characterized by a lysosomal accumulation of unesterified cholesterol and other lipids in many cell types, probably due to impairment of the retrograde transport of lipids from the late endosomes and/or lysosomes to the plasma membrane or endoplasmic reticulum (ER). The clinical manifestation includes hepatosplenomegaly, vertical gaze palsy, and progressive neurodegeneration characterized by cerebellar ataxia, bulbar dysfunction, and variable degrees of cognitive decline. Most often, the onset of symptoms occurs in early childhood, leading to death within a decade. Two human genes have been identified for NPCD: mutations in NPC1 are causative in nearly 95% of all NPC cases, while mutations in NPC2 account for the rest. Over 230 mutations have been identified in the NPC1 gene, nearly ⅔ of which are missense mutations. NPC1 is a highly-conserved integral membrane protein with 13 transmembrane domains, while the NPC2 gene encodes a small soluble protein. Both proteins are located in late endosomes and lysosomes. Recent structure and biochemical studies have revealed that NPC1 and NPC2 bind to cholesterol in opposite orientations, leading to a working model of these two proteins: NPC2 captures the cholesterol liberated from LDL, and then transfers it to the NPC1 on the membrane for subsequent trafficking out of the lysosomes. In NPCD patients, the cholesterol trafficking out of late endosomes and lysosomes is blocked resulting in an accumulation of free cholesterols in late endosomes and lysosomes. This accumulation of cholesterol eventually affects the lysosomal homeostasis, triggering also the accumulation of other lysosomal substrates such us glycosphingolipids. Moreover, glycosphingolipids accumulation in sphingolipid storage diseases (SLSD) as result of a defective lysosomal hydrolase or activator protein also affects cholesterol homeostasis elevating cellular cholesterol levels.
Currently, there is no cure for NPCD and all established therapies are for relief of symptoms with limited efficacy. Clinical trials with Miglustat (N-butyldeoxynojirimycin, Zavesca®) are in progress with favorable preliminary results. Miglustat is an iminosugar that inhibits glucosylceramide synthase, an enzyme responsible for a series of reactions that lead to the synthesis of most glycosphingolipids (GSL). This drug crosses the blood-brain barrier, reduces the substrate availability for synthesizing GSL, and thus reduces GSL accumulation in the brain. This substrate reduction effect seems to relieve the symptoms in NPC patients. Recently, several other compounds including allopregnanolone, T0901317, curcumin and cyclodextrin have also been reported to have beneficial effects on NPC cell or animal models. The full therapeutic benefits of these drugs still need to be evaluated. Despite these studies, an effective treatment for NPC patients is still an unmet medical need.
Recent advances in technologies for high-throughput screening (HTS) have made it possible to screen the cell-based NPC disease model against compound libraries to identify lead compounds for the new drug development. Two such attempts have been made which applied the filipin cholesterol staining assay in NPC patient-derived skin fibroblasts. Though some active compounds were reported from these screens, none of them are useful for the further drug development. In addition, the drug development process usually takes an average of 8-12 years costing on the order of hundreds of millions of dollars. The failure rate of drug development is very high due to unpredictable compound toxicity in human and disproportional efficacy between human and model systems. The present invention is directed toward overcoming the problems discussed above.